| ObjectiveAtherosclerosis mice model were induced by high fat diet-fed of apolipoprotein E（ApoE）knockout mice,explore the traditional Chinese medicine（TCM）syndrome attributes of the model,to research the relationship between AS and gut microbiota and bile acid metabolism,as well as the intervention effect of Xie Xin Tang（XXT）and the corresponding molecular mechanism,so as to provide objective basis for the application of heat-clearing and detoxification method in AS.Methods1.Theoretical discussion:By reviewing relevant domestic and foreign literature,combing the systemic understanding of AS in TCM,clarifying the importance of endogenous hot poison mechanism in the formation of AS,discussing the research on the treatment of cardiovascular disease by heat-clearing and detoxification method,and the intervention of XXT research.Analyze the relationship between AS and lipid metabolism,inflammation,gut microbiota,bile acids from western medicine.2.Experimental research:SPF male C57BL/6J mice:control group,control group plus Xie Xin Tang group;and ApoE-/-mice were randomly divided into 2 groups:model group,model group plus Xie Xin Tang group.AS mice model were induced by high fat diet-fed of ApoE-/-mice.The mice were fed with deionized water and XXT daily for 12weeks;the mice’s body weight,water intake and food intake were monitored;the 12th week of the experiment,collected feces sample.After the administration,fasting,anesthetized,collected blood,sacrificed mice,and took the tissues including aorta,gallbladder,liver,large intestine,cecum feces,etc.HE staining of the aorta and large intestine tissues was used to observe the changes of tissue morphology and structure;determine blood lipid levels;detect the expression of TNF-αand IL-1βin plasma by ELISA.observe mucin in the large intestine tissue by using Alcian blue and WGA staining;The 16S r RNA technology was used to sequence cecum feces;RT-PCR were detected the expression of Occludin m RNA in large intestine tissue,Cyp7b1,Cyp8b1,Lxr,and Fxr m RNA in liver tissue;and the protein expression of FXR,TGR5,AMPK,P38 and JNK in liver tissue were detected by Western blot.LC-MS technology to analysis fecal bile acids composition.Results1.General situation:The mice in control group were in good mental state,drinking and eating well,and their hair was bright.Some mice in model group showed different degrees of fidgety,thirsty and fond of drinking,dry stool or constipation.After XXT intervention,the mice were fidgety,thirsty,constipation and other symptoms were improved.2.Body weight,water and food intake,feces morphology:Compared with control group,in model group the body weight of mice gained relatively faster,water intake was significantly increased（P<0.01）,and food intake was no significant changed,the feces of mice wete small and dry.Compared with model group,in XXT group the body weight of mice increased slower（P<0.05）,their water intake was significantly decreased（P<0.01）,there was no significant difference in food intake;the symptoms of feces were slightly relieved.3.HE staining of aorta:In control group,the aortic intima surface of mice was smooth,and endothelial cells were arranged in order,and no atherosclerotic plaque formation.In model group,the aortic intima was thickened,and there were foam-like cells under the aorta intima,and the plaque area was significantly larger than other groups.In XXT group,the damage on the aortic intima surface was reduced,only a few foam-like cells were found,and the plaque area was reduced.4.Lipid levels:Compared with control group,the plasma TG,TC and LDL-C levels of model group were significantly increased（P<0.01）.Compared with model group,the TC and LDL-C levels of XXT group were significantly decreased（P<0.05）;the TG level was reduced but there was no significant difference.5.ELISA assay:Compared with control group,in model group the plasma pro-inflammatory cytokines of TNF-αand IL-1βwere significantly increased（P<0.01）.Compared with model group,the TNF-αwas significantly reduced in XXT group（P<0.01）,and IL-1βwas also reduced but there was no significant difference.6.Large intestine staining:（1）HE staining:In control group,the large intestine tissue structure was complete,the intestinal muscularis was evenly distributed,and the mucosa layer contained a large number of goblet cells.In model group,the large intestine tissue was damaged,the intestinal muscularis became thinner,and the goblet cells of mucosa layer was reduced.After XXT intervention,the intestinal muscularis recovered and the goblet cells in the mucosa layer increased.（2）Alcian blue and WGA staining:In control group,the intestinal goblet cells of the mice have a complete structure and the intestinal mucin secretion was normal.In model group,the goblet cells of the large intestine tissue were damaged and the mucin secretion decreased.The secretion of intestinal mucin was increased after XXT treatment.7.Gut microbiota analysis:（1）αdiversity:Compared with control group,αdiversity index of Chao1,Observed species,PD whole tree and Shannon were significantly decreased in model group（P<0.01）;αdiversity index increased significantly after XXT treatment（P<0.05）.（2）Gut dominant microbiota at genus level:In control group,the bacteria with higher genus abundance was Akkermansia;In model group,the genus abundance of Faecalibaculum,Bifidobacterium,and Coriobacteriaceae＿UCG-002 were higher.In control group plus XXT group,the bacteria with higher genus abundance were Ruminococcaceae＿UCG-014,Eubacterium＿fissicatena＿group,Bacteroides,Prevotellaceae＿UCG-001 and Alloprevotella.In model group plus XXT group,Lachnospiraceae＿NK4A136＿group,Desulfovibrio and Eubacterium＿Xylanophilum＿group showed higher genus abundance.8.RT-PCR results:Compared with control group,the m RNA levels of Occludin in large intestine tissue and Cyp7b1,Cyp8b1,Lxr,Fxr in liver tissue were significantly down-regulated in model group（P<0.01）.After XXT administration,the m RNA levels of Occludin,Cyp8b1,Lxr were significantly up-regulated（P<0.05）,and the m RNA levels of Fxr,Cyp7b1 were up-regulated,but there was no statistical difference.9.Western blot analysis:Compared with control group,in model group the total protein levels of TGR5,AMPK in liver tissue were significantly down-regulated（P<0.05）,the protein levels of p-p38 was significantly up-regulated（P<0.01）,FXR was down-regulated and p-JNK was up-regulated,there was no statistical difference.Compared with model group,after XXT treatment the protein level of p-P38 was significantly down-regulated（P<0.01）,AMPK was up-regulated,and p-JNK was down-regulated,there was no significant difference.The TGR5 and FXR protein levels were no obviously changed.10.Fecal bile acids metabolite:Compared with control group,the total fecal bile acids,primary bile acids and secondary bile acids were significantly increased in model group（P<0.05）.After XXT administration,the total fecal bile acids and secondary bile acids were increased,but there was no statistical difference.In addition,the levels of UDCA and TCDCA in primary bile acids were significantly reduced after XXT administration（P<0.05）.11.Correlation analysis:The results showed that the beneficial bacteria,such as Akkermansia,Bacteroides,Lactobacillus were negatively correlated with the lipid levels（TG,TC,LDL-C）,pro-inflammatory cytokines（TNF-α,IL-1β）,primary bile acids and secondary bile acids.There were positively correlated with the m RNA levels of Occludin in large intestine tissue and Cyp7b1,Cyp8b1,Lxr,Fxr in liver tissue.However,Anaerotruncus,Mucispirillum and other bacteria to be contrary to above results.Conclusion1.AS mice model were induced by high fat diet-fed of ApoE-/-mice,which reflected the part symptoms of heat syndrome in TCM.XXT could regulate the body weight gain,thirst,and dry stool in AS model mice.It was speculated that high fat diet-fed ApoE-/-mice induced AS animal model was related to heat syndrome in TCM.2.XXT could improve the damage of arterial intima,regulate the lipid levels,inhibit the pro-inflammatory cytokines,and inhibit the formation of AS plaques.3.XXT could protect intestinal barrier,increase the diversity and richness of gut microbiota,regulate the gut microbiota disorder to some extent.Therefore,the improvement of gut microbiota disorder may be one of the mechanisms of XXT inhibited the formation of AS plaque.4.XXT could promote the synthesis of bile acids to a certain extent,increase the size of bile acids pool.So regulated bile acid metabolism abnormal may be one of the mechanisms of XXT inhibit the formation of AS plaque5.The effects of XXT could improve the lipid levels,pro-inflammatory cytokines,gut barrier impaired and bile acid metabolism abnormal in AS model mice may be related to gut microbiota changed. |
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